Epicardial fat density, coronary artery disease and inflammation in people living with HIV

Studies have shown an increased risk of coronary artery disease (CAD) in the human immunodeficiency virus (HIV) population. Epicardial fat (EF) quality may be linked to this increased risk. In our study, we evaluated the associations between EF density, a qualitative characteristic of fat, and inflammatory markers, cardiovascular risk factors, HIV-related parameters, and CAD. Our study was cross-sectional, nested in the Canadian HIV and Aging Cohort Study, a large prospective cohort that includes participants living with HIV (PLHIV) and healthy controls. Participants underwent cardiac computed tomography angiography to measure volume and density of EF, coronary artery calcium score, coronary plaque, and low attenuation plaque volume. Association between EF density, cardiovascular risk factors, HIV parameters, and CAD were evaluated using adjusted regression analysis. A total of 177 PLHIV and 83 healthy controls were included in this study. EF density was similar between the two groups (−77.4 ± 5.6 HU for PLHIV and −77.0 ± 5.6 HU for uninfected controls, P = .162). Multivariable models showed positive association between EF density and coronary calcium score (odds ratio, 1.07, P = .023). Among the soluble biomarkers measured in our study, adjusted analyses showed that IL2Rα, tumor necrosis factor alpha and luteizing hormone were significantly associated with EF density. Our study showed that an increase in EF density was associated with a higher coronary calcium score and with inflammatory markers in a population that includes PLHIV.


Introduction
The introduction of highly active antiretroviral therapy in the mid-90s has led to a decrease in the mortality related to human immunodeficiency virus (HIV) infection accompanied by an increase in life expectancy. [1,2] Individuals living with HIV have a higher risk to develop age-related diseases such as coronary artery disease (CAD) and several cohort studies have shown an increased risk of CAD in people living with HIV compared with the general population. [3][4][5] The pathophysiological processes underlying this increased risk are not well understood but may be linked to metabolic changes such as adipose tissue disturbances or inflammation. [6,7] Epicardial fat (EF), which is the visceral fat of the heart, can be considered as an active organ capable of secreting inflammatory cytokines that may have local and systemic effects on cardiovascular disease (CVD). [8,9] Several studies have shown that the amount of EF is increased in participants living with HIV (PLHIV), and that it is associated with a greater risk of CAD. [10][11][12] On the other hand, only a limited number of studies have explored how EF quality, beyond its quantity, can be related to inflammation and cardiovascular health among PLHIV. [13][14][15] Fat quality can be measured using fat computed tomography (CT) density (or CT attenuation, in Hounsfield units [HU]). This qualitative characteristic of fat can vary due to alterations in lipid content, tissue vascularity, and inflammation. [13][14][15][16][17] Studies performed in the general population have shown that changes in fat density may impact CAD risk independently of fat quantity. [13,18,19] In this study, we aimed to measure the associations between EF CT-measured density in both PLHIV and uninfected controls, and levels of inflammatory markers, cardiovascular risk factors, HIV-related parameters, and coronary artery plaque burden.

Study design and study population
We conducted a cross-sectional study nested within the Canadian HIV and Aging Cohort Study (CHACS), an ongoing multicenter prospective cohort following participants with and without HIV, with the aim of identifying the determinants of premature aging in people with HIV. Inclusion and exclusion criteria were previously reported. [20] We report results from the CHACS cardiovascular imaging sub-study, in which consecutive CHACS participants, aged 40 years or older, with a 10-year Framingham risk score between 5% and 20%, without symptoms or history of CAD, with a preserved renal function and without allergy to contrast media were invited to undergo non-contrast cardiac CT and coronary CT angiography. This study was approved by the Institutional Review Board of the Medical Center of the University of Montreal and participating centers. All participants gave written informed consent.

Study procedures
Non-contrast cardiac CT and coronary CT angiography were performed using a 256-slice CT scanner (Brilliance iCT; Philips Healthcare, Best, The Netherlands). Prior to CT, participants were given 50 to 75 mg of metoprolol orally if heart rate was >60 beats/min, and 0.4 mg of nitroglycerin sublingually unless contraindicated.
Non-contrast cardiac CT was performed for coronary calcium scoring and EF evaluation using the following parameters: slice thickness 2.5 mm, matrix 512 × 512, field-of-view 250 mm, scan voltage 120 kV and prospective electrocardiographic-gating. CT angiography was performed for coronary plaque analysis using 370 mg/mL of iopamidol (Bracco Imaging, Milan, Italy) at a flow rate of 5 mL/s. Images were reconstructed using a hybrid iterative reconstruction algorithm (Philips iDose; Philips Healthcare, level 3).

Exposure of interest: EF density
EF is the fat deposit located between the visceral pericardium and the myocardium. EF density decreases with fat gain and increases with fat loss. [16,21] In addition, EF density increases with inflammatory cell infiltration and metabolic dysfunction. [14,16,22] Therefore, the higher the density, the more metabolically active the fat deposition might be. [13,23] EF density and volume were both assessed using non-contrast CT images and a semi-automated software (Aquarius Intuition version 4.4.11; TeraRecon Headquarters, Forster City, CA). The pericardium was manually traced on axial slices from the pulmonary artery bifurcation to the apex of the heart. A CT density between −190 and −30 HU was used to select the EF and exclude any other tissue. Mean EF density and volume were calculated using the software based on the adipose tissue area, the number of slices, slice thickness, and intersection gaps, and reported as a continuous value, expressed in HU and cm 3 .
Two observers both blinded to HIV status and clinical data measured EF volume and density using the semi-automated software. Inter-observer and intra-observer agreement for EF volume and density measurement were highly reproducible (intraclass correlation coefficient for inter-observer agreement 0.75 for EF volume and 0.99 for density; Intra-observer agreement 0.97 for EF volume and 0.97 for density). [24]

Outcome of interest: coronary plaque
Coronary artery calcium (CAC) measurement was performed using the method by Agatston et al [25] on non-contrast CT images. Coronary plaque analysis was performed using CT angiography images as previously described. [26] Plaque volume was quantified in multiplanar reformat. Total plaque, calcified, non-calcified and mixed plaque volumes per participant were defined as the sum of volumes of all plaques, calcified, non-calcified and mixed plaques per participant. Low-attenuation plaque, a marker of plaque vulnerability, was defined as the sum of volumes of all portions of 30 HU or less of each plaque per participant.
Inter-observer and intra-observer agreement for plaque volume analysis was excellent as previously described. [27]

Metabolic and inflammatory biomarkers
Eighty-eight inflammatory, anti-inflammatory and metabolic markers were measured in the CHACS cohort. These biomarkers were measured in plasma collected from the study participants using regular ELISA assays and Meso Scale technology as previously described. [28] In this study, we present results for 11 markers as these were the only one that were significantly associated to EF density in univariable analysis.

Statistical analysis
Continuous variables are reported as mean ± standard deviation or median [25th-75th interquartile range], as appropriate. Categorical variables are reported in frequency and percentages. Student t test or Mann-Whitney U test was used to compare normally and nonnormally distributed variables between participants with HIV and controls and chi-squared test was used to compare categorical variables.
The association of EF density with cardiovascular risk factors, as well as with HIV related parameters, was assessed using univariable and multivariable linear regression models, with and without inclusion of EF volume into the models. Multivariable model included all HIV-related parameters and cardiovascular risk factors risk factors significantly associated with EF density.
The association of EF density with coronary plaque (CAC, total, calcified, noncalcified and mixed plaque volume) was evaluated using zero-inflated Poisson regression, as previously described. [10] The use of zero-inflated model is made necessary by the excess of 0 in the distribution of total coronary plaque volumes. Coronary plaque volume variables were natural-log transformed prior to statistical analysis. Multivariable models were adjusted for CVD risk factors.
In addition and as an exploratory analysis, the association of EF with inflammatory biomarkers was evaluated using univariable and multivariable linear regression models. Multivariable models included inflammatory biomarkers and CVD risk factors if they showed a univariable association with EF. As this analysis was hypothesis-generating, we did multiple tests and did not correct for multiple comparisons.
Effect modification by HIV of each association was assessed by inclusion of an interaction term to the fully adjusted models, and significance of the interaction term was assessed by a likelihood ratio test.
For patients with incomplete data, the mean or median value was used to impute the missing data. Values for the following number of participants were missing and imputed: Body mass index (BMI), [5] smoking exposure, [14] high density lipoprotein-cholesterol, [4] low density lipoprotein-cholesterol, [9] antiretroviral therapy (ART) exposure duration, [7] non-nucleoside reverse transcriptase inhibitors exposure duration. [7] A P value <.05 was considered statistically significant. Statistical analyses were performed using R (version 3.3.2; R Foundation for Statistical Computing, Vienna, Austria).
Subjects with HIV had a mean duration of HIV infection of 18.3 ± 7.7 years. More than 93% of PLHIV were exposed to ART with a mean duration of 13.5 ± 6.5 years. 74.6% were exposed to proteas inhibitors, 93.8% where exposed to nucleoside reverse transcriptase inhibitors, 62.3% were exposed to NNRTIs and 45.2% were exposed to integrase strand transfer inhibitors. Viral load was detectable in 8.5% of PLHIV and mean CD4 count was 611.7 ± 304.0 cells/mm 3 .
All levels of circulating inflammatory markers were similar between the 2 groups except for interleukine-7 (IL7), tumor necrosis factor alpha (TNFα), growth-regulated oncogene alpha and follicular stimulating hormone which were higher in PLHIV compared to controls (all P < .05).

EF density and CVD risk factors
Univariable analysis showed that EF density was negatively associated with age, with fat density decreasing with advancing age (β = −0.13 per 1 year increase in age, P = .004), smoking exposure (β = −0.04 per each additional pack-years of exposure, P = .026), triglyceride (β = −0.80 per 1 mmol/L increase in triglyceride, P = .015), statin use (β = −2.25 for use vs non use, P = .004), BMI (β = −0.32 per 1 unit increase in BMI, P < .001) and EF volume (β = −0.09 for each increase in 1 cm 3 , P < .001) while a positive association was observed with male sex (β = 3.03, P = .003). In a multivariable regression model including all the traditional cardiovascular risk factors and EF volume, only male sex, diabetes and EF volume were independently associated with EF density (Table 3).

EF density and HIV-related parameters
In univariable models, EF density was only significantly associated with NNRTI exposure duration (β = −2.12, per 10 years increase of exposure, P = .031) while no association was observed with other ART classes, HIV duration, CD4 and CD8 count. In a multivariable regression model including all HIVrelated parameters, CVD risk factors, and EF density, none of the HIV related parameters was significantly associated to EF density. Results are presented in Tables 4 and 5.

EF density and coronary plaque burden
Univariable analysis showed no significant association between EF density and coronary calcium score, total plaque volume, subtypes of plaque volume and low-attenuation plaque volume (all P ˃ .05) ( Table 6). Multivariable models adjusting for CVD risk factors showed positive association between EF density and calcium score (logit component odds ratio [OR], 1.07 per 1 HU increase in EF density, P = .023 and OR for Poisson regression = 1.00 per 1 HU increase in EF density, P = .635) while no association was observed with other measures of plaque. Additional adjustment for EF volume showed that the association between EF density and calcium score remained statistically significant (logit component OR, 1.09 per 1 HU increase in EF density, P = .039 and Poisson component OR 1.00 per 1 HU increase in EF density, P = .877) ( Table 6). Table 7 summarizes the results of the association of EF density with metabolic and inflammatory markers among 123 participants with available values (76 HIV+, 47 HIV−). Among the soluble biomarkers measured in our study, univariable regression analyses showed that only IL2Rα, IL7, leptin, peptid C, insulin, TNFα, EAN78, growth-regulated oncogene alpha, follicular stimulating hormone, luteizing hormone (LH) and PP were associated with EF density. After adjustment for CVD risk factors and EF volume, only IL2Rα, TNFα and LH remained significantly associated with EF density.

Main findings of the study
In the current cross-sectional study, we showed that EF density did not differ by HIV status. EF density was positively associated with male sex, diabetes, and coronary calcium score, independent of cardiovascular risk factors and EF volume, but no association was found with HIV specific factors. Finally, we found that EF density was independently associated with some metabolic and inflammatory markers, mostly IL2Rα, TNFα, and LH.

EF density
PLHIV have a higher risk to develop CAD with subsequent complications, as shown in large cohort studies. [3][4][5] Mechanisms underlying this increased risk are not yet fully understood but several factors such as ART, abnormal fat deposition, inflammation or immune activation may play a role.
HIV infection and use of ART can lead to alterations in fat distribution. [29,30] We and others have previously showed that Medicine quantity of EF was increased in PLHIV compared to uninfected controls. [10,11,31] Studies in the general population as well as in PLHIV have demonstrated that greater quantity of EF was associated with a greater coronary atherosclerotic plaque burden. [11,32,33] EF secretes bioactive cytokines that are known to promote atherosclerosis. [8,34]  18.3 ± 7.7 Participants exposed to ART, n (%) 166 (93.8%) ART exposure duration (yr)* 13.5 ± 6.5 Participants exposed to PIs, n (%) 132 (74.6%) PIs exposure duration (yr)* 9.5 ± 5.1 Participants exposed to NRTIs, n (%) 166  Recently, assessment of EF quality has gained interest. However, only a few studies reported results specific to the HIV population.

EF density and HIV infection
Unexpectedly, PLHIV in our cohort did not have a significantly different EF density compared to healthy controls, despite having a higher volume of EF. As in our study, Buggey et al, [35] did not find a significant difference between HIV-positive and HIV-negative participants when comparing EF density measured in the left atrium roof and peri-right coronary artery. In another study evaluating density of abdominal visceral fat, fat density did not differ by HIV status. [36] EF inflammation and fibrosis may had alleviated the expected decrease in fat density and therefore explain the absence of difference in fat density between HIV-positive and HIV-negative participants despite the difference in EF volume.
In our study, we observed an inverse correlation between EF density and NNRTI exposure duration and positive association with detectable viral load in univariable analyses. However, these associations did not persist after adjustment for CVD risk factors and EF volume. In their study, Longenecker et al [37] found that baseline EF density and changes over time were inversely correlated with ART and duration of protease inhibitor exposure. Similarly, studies evaluating abdominal visceral fat demonstrated that exposure to ART was associated with a lower density of fat. [16,38,39] We and others have demonstrated that a longer exposure to ART and to some specific ART classes were associated with an increased EF volume. [10,11]

EF density and coronary plaque burden
To our knowledge, our study is the first to evaluate the association of EF density with coronary artery plaque burden in PLHIV. We found that EF density was positively associated with coronary calcium score in multivariable models adjusting for CVD risk factors and EF volume.
Our results are consistent with findings in the general population. In their studies, Pracon et al [40] and Liu et al [18] showed  Beta is interpreted as the mean increase in epicardial fat attenuation (in HU) predicted by the model per each one unit change in the explanatory variable. A negative beta coefficient indicates a decrease. Medicine that EF density was positively correlated to coronary calcium score. Other studies demonstrated a positive association of EF density with coronary atherosclerosis severity as well as with myocardial infarction. [13,18,41] Higher EF radiodensity may reflect fat inflammation and fibrosis and represent a form of unfavorable metabolic activity which may directly affect coronary atherosclerosis. The association of EF density with coronary plaque burden was independent of EF volume suggesting that different mechanisms may account for clinical effect. Of note, some groups have reported opposite association between EF density and coronary plaque and showed that decreased density was associated with impaired CAD profile. [42][43][44] This may be explained by the inclusion of patients with different degrees of inflammation and fibrosis in EF.
Future mechanistic studies will need to be carefully designed to unravel the complex relationship between fat quality and CAD.

EF density and metabolic and inflammatory biomarkers
Fat abnormalities in PLHIV have been associated with metabolic dysregulation, inflammation, and immune activation. In our study, we evaluated a selected panel of metabolic and inflammatory markers and showed that only IL2Rα, TNFα and LH were positively associated with EF density independent of CVD risk factors and EF volume. IL2 receptor subunit alpha plays an important role in the proliferation and differentiation of T cells. [45,46] High levels of IL2Rα have been associated with multiple inflammatory diseases such as rheumatoid arthritis, [47] as well as with CAD. [48] TNFα represents one of the most potent pro-inflammatory cytokines. Studies have demonstrated that TNF-α has roles in the development of atherosclerosis. [49,50] Finally, recent findings demonstrated that adipose tissue regulates the LH secretion via leptin production. [51] Elevated levels of LH are associated with abnormalities predisposing to CVD risk such as insulin resistance, dyslipidemia, and systemic inflammation. [52,53] Our findings support the hypothesis that an abnormal EF quality may be linked to subclinical atherosclerosis through its underlying source of inflammatory mediators potentially favoring atherosclerosis. Consequently, CT measurements of quality of EF might add valuable information in the cardio-vascular risk assessment of HIV individuals.
Only few studies evaluated the correlation of fat density with inflammatory markers in PLHIV and found results comparable Table 4 Association of HIV specific variables with epicardial fat density in participants living with HIV, N = 177 (including overall antiretroviral therapy exposition duration). Defined as viral load greater than 40 copies/mL (0: no detectable viral load, 1: detectable viral load). Table 5 Association of epicardial fat density with HIV-specific variables in participants living with HIV, N = 177 (including antiretroviral therapy class exposition duration). to ours. Longenecker et al, [37] and Chen et al, [54] showed that density of EF was negatively correlated to hs-CRP, IL6 and positively correlated T-cell activation. Similarly, other studies evaluating abdominal visceral fat density showed that it was negatively correlated to hs-CRP, IL6 and leptin level and positively correlated to adiponectin. [36,38]

Strengths and limitations
Our study has many strengths including its robust design, the use of CT that allowed us to characterize fat radiodensity in addition to volume and coronary plaque burden without additional exposition to radiation. We evaluated EF density in a population of individuals living with HIV and healthy controls, male and female and demonstrated for the first time that EF density was positively correlated to calcium coronary score in a population that includes PLHIV and to metabolic and inflammatory markers independent of EF volume. However, our study did not have sufficient power to assess whether this link was specific to PLHIV. There are several limitations of our study: First, there may be significant regional variation of adipose tissue characteristics, particularly around atherosclerotic plaques. Future studies in the HIV population should consider more granular characterization of radiodensity by location. As we have previously reported, our study was conducted in participants who were predominantly male. It remains unknown whether these findings can be generalized to women. Although this does not bias our results, it limits their generalizability. Finally, this was a cross-sectional analysis which does not allow for determination  of causality. Additionally, as a small group of participants had calcified, noncalcified or mixed plaques, the analyses on plaque sub-types may have lacked statistical power. This was also true for antiretroviral therapy, where a small group of participants were in each sub-class of ART. We performed a fair number of statistical tests, especially for the association of EF density and metabolic and inflammatory markers and we chose not to adjust p values for multiple comparisons which may have increase the likelihood of obtaining false positive results. But this was a hypothesis-generating study and further studies are needed to confirm our findings. As PLHIV live longer, understanding the relationship between HIV, ART, and contributors to CAD risk is important. Our study shows that an increase in EF density is associated with a higher coronary calcium score in a population including PLHIV. An increase in EF density is also associated to metabolic and inflammatory dysfunction. Future measurement of EF density may provide additional insight into adipose tissue function beyond measurement of quantity alone and may have implications for assessment of long-term cardiovascular risk.